Cytokine receptor peptides, compositions thereof and methods thereof

ABSTRACT

The invention provides a pharmaceutical composition including a peptide comprising at least a portion of a chemokine receptor or a G-protein coupled receptor and optionally a cytokine. The pharmaceutical composition of the invention may be used for altering immune system functioning, for example, to treat an immune system disorder, such as an autoimmune disease, multiple sclerosis, transplant rejection, psoriasis and asthma. The invention also provides peptides that may be used in the pharmaceutical composition and a method for preparing the pharmaceutical composition of the invention. The invention further provides a method for to treating an immune system disorder, such as an autoimmune disease, multiple sclerosis, transplant rejection, and psoriasis asthma.

CROSS-REFERENCE TO EARLIER APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/225,122, filed Sep. 8, 2009, now allowed, which is a National PhaseApplication filed under 35 U.S.C. 371 as a national stage ofPCT/IL2007/000350, filed Mar. 18, 2007, and application claiming thebenefit under 35 U.S.C. 119(e) of U.S. Provisional Application No.60/782,689, filed Mar. 16, 2006, each of the above applications beinghereby expressly incorporated by reference in its entirety and eachbeing assigned to the assignee hereof.

FIELD OF THE INVENTION

This invention relates to peptides which bind cytokines and their use inimmune disorders.

BACKGROUND OF THE INVENTION

The following publications are considered relevant for an understandingof the invention.

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Chemokines in Health and Disease

The immune system promotes health by combating foreign pathogens,alleviating intrinsic disease and repairing physical injury. Infected,injured, or otherwise compromised tissues mobilize cells of the immunesystem by releasing chemo-attractants called chemokines (CKs) into theblood stream. The same CKs induce the activation of leukocytes anddirect the differentiation of lymphocytes [Rossi, 2000; Zlotnik, 2000].The deployment of immune cells is essential, not only to confrontpathogenic challenge, but also for immune surveillance and tolerance to“self” [Mackay, 2001]. Close to fifty different human CKs and twenty CKreceptors (CKRs) respond to multifarious pathogens and disease states.Tight regulatory control of the CK system imparts rapid, measured andapposite responses to the various pathogenic challenges. Of equalimportance to health is the control which prevents the immune systemfrom acting against “self”. An immune response which is inappropriate,excessive, or protracted, relative to the pathogenic insult, if any,will cause injury to healthy tissue. Such aberrant immune responses areresponsible for the clinical conditions of multiple sclerosis,inflammatory bowel disease (Crohn's disease, ulcerative colitis),rheumatoid arthritis, psoriasis, asthma and juvenile diabetes, the majorand prevalent autoimmune diseases. Experimental studies with animalmodels of disease [Gerard, 2001; Dogan, 2004; Szekanecz, 2003] andclinical observations [Gerard, 2001; Godessart, 2001] indicated that thelevels of CKs and their cognate receptors correlate with specificautoimmune diseases.

Current Treatment of Autoimmune Disease

The etiology of no single autoimmune disease is known, but diseasepathology, in every case, is the result of immune cell activity directedagainst “self”. It follows, therefore, that the “state of the art”treatment is restricted to the induction and maintenance of diseaseremission. Multiple sclerosis (MS), the most common cause ofneurological disability in young adults, presents with arelapsing-remitting course and is followed by a secondary progressivephase. Steroids are the first choice of treatment to shorten theduration of relapse and accelerate recovery. Long term treatment, withNon Steroidal Anti Inflammatory Drugs (NSAIDs), is given to maintain, orextend remission. Inflammatory bowel disease (IBD) affects over 1million Americans today. Manifested by severe abdominal pain anddiarrhea, it is associated with an increased risk of bowel cancer. Forpatients suffering mild IBD, remission is induced with NSAIDs. Becauseof the adverse side effects of steroids, they are reserved for patientswith moderate to severe disease and for those who do not respond toNSAIDs. When the disease is refractory to steroids, immunosuppressantdrugs are used.

Established and new treatments of autoimmunity act by modulating and ifnecessary, suppressing the immune system. Both approaches run the riskof side effects, the short term of which are known, the long term, whichcannot always be predicted. The shortcomings of steroids, the first lineof treatment for severe MS and IBD are well documented:gastrointestinal, dermatologic, neurological, endocrinological,ophthalmic and metabolic side effects. Remission in MS is “extended” byinterferon beta and glatiramer acetate (Copaxone), but drug efficacy,tantamount to a 30% reduction in the frequency of relapse, is offset bysevere side effects, such as fatigue, pain and bladder dysfunction. Therange of medications extending remission in IBD is wider, but the sideeffects no less severe. NSIADs are intolerable to a significant numberof IBD patients and the alternatives, the immuno-suppressants 5-AZT and6-mp, cause severe side effects. Prolonged use of a new generation ofantibody drugs, exemplified by Remicade, is beginning to reveal anegative side. Remicade has been linked with tuberculosis, opportunisticinfection and the activation of latent MS (Centocor, Inc. 2005). Add tothis deteriorating safety profile the high cost and administration byintravenous infusion and this innovative treatment becomes a less thanan attractive alternative to tried and tested medications. There is aneed for improved medication to treat acute inflammation in diseaserelapse and provide long term treatment to maintain remission.

Chemokine Receptors (CKRs) as Conventional Drug Targets

The CKR, a member of the G-protein Coupled Receptor (GPCR) family, has adrug target pedigree. More than forty five percent of all marketed drugstarget GPCRs [Horuk, 2003]. Given that the ligands of GPCRs are lowmolecular weight peptides (histamine, dopamine, serotonin), it waspredicted that CKRs would be tractable targets for small molecule drugs(Proudfoot et al., 2003). The majority of drug candidates targetingCKRs, including those under development and those which have beenabandoned, are indeed small molecule inhibitors [Wells, 2006]. Based onsteric and energetic considerations [Onuffer, 2002] and empiricalstudies [Sabroe, 2000], it was proposed that small molecule antagonistsof CKRs should be non-competitive inhibitors of CKs that blockligand-receptor interaction by stabilizing the receptor in an inactiveconformation.

CKRs are known to interact with both their cognate and with unrelated CKligands, a phenomenon known as “redundancy”. As a functional definitionof CK-CKR interaction, a “cognate” CKR-CK pair is defined as a CKR and aCK for which the CKR is activated by nanograms of the CK to induceintracellular signaling (calcium mobilization, kinase and lipaseactivation). With respect to the same CK-CKR system, an “unrelated”CKR-CK combination is defined as a CKR which requires at least threeorders of magnitude more of CK, micrograms, to elicit a positive,negative, or unproductive intracellular response.

The identity of CKRs as GPCRs is an evident advantage for drugdevelopment. The atypical involvement of these GPCRs in physiologicalimmunity, however, makes CKRs equivocal drug targets and even atherapeutic liability. Antibodies against the receptor, CCR2, which isexpressed by inflammatory T cells and monocytes, were used to treatexperimental collagen-induced arthritis [Bruhl, 2004]. The treatment wastherapeutic during disease initiation (day 0-15), but deleterious indisease progression (day 21-36). It transpired that a sub-population ofregulatory T cells, responsible for immune tolerance, is expandedseveral fold during the phase of disease progression. The antibodies, byblocking the CCR2 receptors of regulatory T cells, exacerbated diseasesymptoms Inhibition of the same drug target expressed by different celltypes, CCR2 in the example, can be therapeutic, or pathologic, dependingon the function of the cell expressing the targeted CKR. The CK,RANTES/CCL5* (*CK classification: [Zlotnik, 2000]) and its cognatereceptor, CCR5, are examples of disease-related proteins that are alsoessential for physiological immunity. Elevated levels of CCL5 and CCR5correlated with glomerular cell proliferation and macrophageinfiltration in experimental glomerulonephritis. CK analogues(Met-RANTES and amino-oxypentane-RANTES), which block the receptor,reduced glomerular cell proliferation and macrophage infiltration, butaggravated clinical symptoms [Anders, 2003]. The CK analogues wereobserved to be therapeutic antagonists of leukocyte recruitment, butpathogenic activators of resident macrophages. CKRs, validated GPCR drugtargets, are potential therapeutic liabilities as mediators ofphysiological immunity.

CKs and CKRs constitute a network of interacting proteins [Schwarz,2002]. Drugs directed at individual proteins of a network riskperturbing the network as a whole. It follows, therefore, that wheninteracting and interdependent proteins of the network are essential forphysiological immunity, targeting one of the proteins may affect theoverall function of the immune system and so reduce the drug's efficacy.CK gene knock-out has been the preferred experimental approach to studythe physiological consequences of CK network interference. In acomparative study of inflammatory cells from MCP1/CCL2 null mice andtheir wild type counterparts, it was found that eliminating MCP-1/CCL2altered immune responses to disease [Ferreira, 2005]. Equivalent tissuesand cells from null and wild-type diseased mice showed significantdifferences in their respective CK and CK receptor profiles. Networkprinciples were also observed in tissues derived from the MCP-1/CCL2null mice. Reconstituted expression of MCP-1/CCL2 in macrophages of theCK knock-out mice selectively suppressed the expression of CKs, thosespecifically induced by MCP-1/CCL2 inactivation. Physiological immunityin genetically altered animals, as in their wild type counterparts, wasdictated by principles of network responses and adaptation.

In experimental animal models of disease, the relevance of networking ismost conspicuous at the cellular level. Experimental autoimmuneencephalomyelitis (EAE), a rodent model of multiple sclerosis, was usedto study the role of CCR2 in disease development [Gaupp, 2003]. Therecruitment of monocytes and macrophages into the CNS was examined inCCR2 knock-out, disease-induced mice. Contrary to what was expected,inactivation of CCR2 did not confer resistance to EAE in the mice. CNSlesions did contain diminished levels of monocytes, proof of impairedmonocyte function, but also elevated levels of neutrophils. CK(IL-8/CXCL8) and CK receptor (CCR1, CCR5) profiles of the experimentalmice were found to be consistent with augmented neutrophil levels in thelesions. It was proposed that the CK-CKR network of the CCR2 null miceresponded to disease induction with a “compensatory” immune responseinvolving alternative immune regulator molecules and effector cells. Theforegoing example is yet another expression of the network principle.Neutralization of a disease-related protein, one which is important forphysiological processes in general, can elicit a counter response, whichitself can be pathological. A murine model of experimental arthritis,which reproduces the symptoms of severe human rheumatoid arthritis,provided another illustration of the physiological consequences ofnetwork interference [Quinones, 2004]. CCR2 knock-out mice were used tostudy the role of the receptor in experimental arthritis. The CKR isimplicated in disease pathogenesis, therefore, it was not anticipatedthat the phenotype of the CCR2 knock-out mice would be similar to thatfound in severe human arthritis, elevated T cell levels and monocytesand macrophages concentrated in the inflamed joints. The profferedexplanation was that disease in the CKR compromised mice stimulated theexpression of alternative CKRs to mobilize the inflammatory cells. Theconsequence of perturbing a physiologically essential network, even fortherapeutic effect, can be to elicit compensatory reactions toconsolidate, but potentially exacerbate, the status quo.

The CK-CKR axis, despite the “druggable” attributes of GPCRs and theirsmall molecule ligands, is demonstrably problematic as a drug target. Itfunctions as a network of interactive and interdependent regulatoryproteins central to physiological immunity. Compensatory and possiblydeleterious responses may therefore be the unavoidable consequences ofpharmacological intervention in the network.

The Chemokine-Chemokine Receptor Network

A disproportionate number of chemokine (CK) ligands, ill-defined ligandspecificity and equivocal CK functionality in vitro, were cited as proofof CK “redundancy” and worse still, “promiscuity”. Such epithets,although consistent with data at the time, were incompatible with aCK-Chemokine Receptor (CKR) network essential for physiological immunityand pathological autoimmunity. Re-examination of the original data, inthe light of subsequent functional studies and structure-activityanalyses, reveals physiological relevance in both the ligand to receptorratio and ostensibly indiscriminate receptor activation. To begin with,CKs, which are agonists of their cognate receptors, were found to benatural antagonists of unrelated receptors, as determined by chemotaxisand Ca²⁺ flux assays. Three agonists of the receptor, CXCR3, MIG/CXCL9,IP10/CXCL10 and I-TAC/CXCL-11, were shown to be antagonists of theunrelated receptor, CCR3 [Loetscher, 2001]. The most potent of thethree, I-TAC/CXCL11, was found to be a natural antagonist of CCR5[Petkovic, 2004]. Eotaxin/CCL11, an agonist of CCR3, was also shown tobe an agonist and natural antagonist of the unrelated receptors CCR5 andCCR2, respectively [Ogilvie, 2001]. Another cognate ligand and agonistof CCR3, Eotaxin3/CCL26, was found to be a natural antagonist ofunrelated receptors CCR1 and CCR5 [Petkovic, 2004]. MCP3/CCL7, a cognateagonist of receptors CCR1, 2 and 3, was shown to be a natural antagonistof the receptor, CCR5 [Blanpain, 1999]. The concomitant activation andinhibition of CKRs was interpreted to be a mechanism for regulating therecruitment of functionally discrete sub-populations of leukocytes. Toillustrate the point, MIG/CXCL9 and Eotaxin/CCL11, by altering thebalance between Th1 and Th2 cells, could determine the course ofAllergic Airway Inflammation [Thomas, 2004; Fulkerson, 2004]. MIG/CXCL9is an agonist of CXCR3, expressed by Th1 cells and an antagonist ofCCR3, expressed by Th2 cells. Eotaxin/CCL11, a cognate ligand of CCR3,was also shown to have a high affinity binding site in the unrelatedreceptor, CXCR3 [Xanthou, 2003]. A refinement of the point,activation/inhibition of CKRs, was regulation of T cell recruitment bydifferential activation of the same receptor [Thomas, 2002].IP-10/CXCL10 and MIG/CXCL9, cognate ligands of CXCR3, could respectivelyenhance, or diminish eosinophil accumulation and airwayhyper-reactivity. Cumulative data now show that the disease-relateddistribution and activities of immune cells are in fact a corollary ofCK multiplicity and specificity for diverse CKRs.

Since neither experimental data, nor clinical evidence distinguishesbetween wild type and disease-related CKs, it is presumably the samechemo attractants that mobilize the immune cells of physiological andpathological immune responses. CK pathogenicity appears to be theconsequence of inordinate, inappropriate and enduring wild-type CKactivity, phenomena consistent with CK network deregulation. CK toreceptor binding analyses were carried out to elucidate the molecularbases for the regulation of CK-CKR interactions. MIG/CXCL9 andIP-10/CXCL10 bind competitively the receptor CXCR3 but non-competitivelywith respect to the third related CK, ITAC/CXCL11 [Cox, 2001]. In thisexample, functionally related CKs were shown to interact with discreteand overlapping sites in their cognate receptor. The anomalousactivities of CKs, those of CKs interacting with unrelated receptors,may be more informative about CK network regulation per se. For example,the cognate ligands of CXCR3 inhibited CCR3 functional responses andwere observed to displace that receptor's cognate ligand, Eotaxin/CCL11[Loetscher, 2001]. A more detailed study of the same system disclosedthat ITAC/CXCL11, cognate ligand of CXCR3, efficiently displacedEotaxin/CCL11 from the extra-cellular loops of the latter's receptor,CCR3. Cognate and unrelated ligands, therefore, can share overlappingbinding sites within the same receptor [Xanthou, 2003]. Anotheranomalous, but informative observation, concerns Eotaxin/CCL11 and itsunrelated receptor, CXCR3. Although Eotaxin/CCL11 is neither an agonist,nor an antagonist of CXCR3 in vitro, the receptor has a high-affinitybinding site for this CK which can be competitively occupied byITAC/CXCL11 [Xanthou, 2003].

Chemokines as Innovative Drug Targets

An upshot of the molecular structure-activity analyses and bindingstudies is that receptors comprise CK binding sequences that, given thecontext, are regulators of CK activity. In the context of the CKR,regulatory elements are potential, but problematic drug targets, becausethe receptors are essential for physiological immunity.

Functional studies and complementary molecular analyses of CK receptors,have disclosed regulatory sequences in the receptors of physiologicaland disease-related CKs. Anomalous interactions of CKs with unrelatedreceptors implicate ubiquitous cryptic regulatory sequences importantfor general CK-CKR network activity. CKR derived CBPs, capable ofmodifying CK binding activities, are not without precedent. In a studyto identify and define receptor sequences responsible for IL-8/CXCL8 andGRO-α/CXCL1 binding, receptor-derived sequences were shown to becompetitive inhibitors of CK binding [Gayle, 1993]. A peptide derivedfrom an extra-cellular domain of the same receptor, CXCR1, was found tobe an antagonist of the cognate CK, IL8/CXCL8 [Attwood, 1996] and whenchemically modified, the peptide was made a stronger inhibitor [Attwood,1997]. More recently, an MCP1/CCL2 binding peptide, homologous to asequence in an extra-cellular loop of the CKRs CCR2 and CCR3, was shownto be angiostatic by antagonizing MCP-1/CCL2 binding to CCR2 [Kiln,2005].

Mechanism of Drug Action—Redress Of Immune Imbalance

The pathogenesis of all autoimmune diseases is patently de-regulated anddysfunctional immune activity. A challenge for any therapeutic approachis to manipulate immune processes that, in the same individual, bothcause clinical symptoms and protect against disease. To date, no therapyhas met the challenge satisfactorily. Autoimmune diseases are treatedwith low efficacy drugs which cause significant side effects. Most ofthe current drugs are small synthetic molecules whose deficienciesappear to derive from their mechanism of action, irreversible inhibitionof disease related proteins.

Drug candidates targeting CKRs belong to the class of small syntheticmolecules [Wells, 2006]. Following from the preceding discussion, drugsof this type are inherently flawed. None can be expected to block andneutralize a CKR with impunity, given the organization of CKs and theirreceptors as a network which is essential for physiological immunity.The principles of network activity govern immune responses at themolecular and cellular levels and dictate the outcome, therapeutic, ordeleterious, of inhibitory drug activity. The overriding objective ofthe network is to maintain immune balance, any perturbation of thenetwork eliciting a response to redress the balance. Experimental modelsof autoimmune disease and clinical evidence provide manifold examples ofnetwork activity where disease manifests itself as immune imbalance andhealth is restored by redressing the balance. Take, for example,allergen challenge in the lung, which elicits deleterious eosinophilsand protective Th1 lymphocytes [Fulkerson, 2004; Thomas, 2002].Allergen-induced CK, Eotaxin/CCL11, recruits inflammatory eosinophils.At the same time, Th1 cells express the CK, MIG/CXCL9, a naturalinhibitor of eosinophils. The inference was that the inflammatory statusof the allergic lung was dictated by competition between positive andnegative regulatory CKs, the result of which competition was translatedinto a balance between Th1 and eosinophilic cells. The clinical symptomsof type 1 diabetes, herpes stromal keratitis and multiple sclerosis, areinfection, tipping of the T helper (Th) lymphocyte balance in favor of atype 1 milieu [Christen, 2004]. Support for the hypothesis was providedby the diabetogenic RIP-LCMV mouse model. Type 1 CKs and cytokines wereshown to be responsible for the diabetic state of the RIP-LCMV mouse. Itwas suggested, with qualification, that redressing the immune imbalanceby inhibiting the type 1 inducing factors, or administering Type 2cytokines, could be therapeutic. Immune balance is argued to be asalient determinant of disease progression in relapsing-remittingmultiple sclerosis [Nakajima, 2004]. Levels of Th1-related CKs areelevated in the active phase of MS whereas MCP-1/CCL2, reported toinduce Th2 reactions, is elevated in remission. An analysis of bloodfrom patients with active MS revealed elevated levels of Th1 lymphocytesand significantly reduced levels of monocytes expressing CCR2, thecognate receptor of MCP-1/CCL2. The results were interpreted as evidencethat elevated MCP1/CCL2 and a subset of peripheral monocytes expressingCCR2, may correct the Th1/Th2 imbalance to create conditions for diseaseremission.

The Th1-Th2 dichotomy influences immune balance in physiologicalimmunity and is a determining factor in causing the immune imbalancethat is characteristic of autoimmune disease. Under prevailingphysiological conditions, Th1 and Th2-like cells are in equilibrium,contributing to the establishment and maintenance of immune balance.Innate and extraneous stimuli induce Th1 and Th2-like immune responsesthat create new, transient equilibria and immune imbalance. Responsesthat entail modification of the immune cell repertoire establish a newTh1-Th2 equilibrium, restoring immune balance. In autoimmune disease, aloss of tolerance is expressed as pathological Th1-Th2 disequilibriumand immune imbalance. In the absence of treatment, resolution of theTh1-Th2 disequilibrium is transient and an enduring immune imbalanceleads to relapse. Effective treatment of an autoimmune disease,therefore, must restore Th1-Th2 cell equilibrium to redress the immuneimbalance for tolerance and disease remission. It follows, therefore,that one way to treat autoimmune disease is to alter the pathogenicequilibrium state. A pharmacological intervention is required to createa new equilibrium and to redress immune imbalance for tolerance. Itsapplication must be incremental and in keeping with the principles ofequilibrium dynamics for maximal therapeutic effect and minimal,detrimental side effects.

A recurrent theme in the study of autoimmune disease is theintercalation of physiological immunity with pathological autoimmunity.Evidence has been presented to show that the former state is achievedwhen immune regulators and effectors are in balance and the latter,disease state, when they are in imbalance. Given that the balance iscontingent on a network of regulatory proteins, negation of a protein,albeit disease related, portends imbalance. Efficacy, therefore, must besought in a drug which modulates and does not negate the disease-relatedactivity of the network-associated protein.

SUMMARY OF THE INVENTION

The present invention is based on the novel and unexpected finding thata combination consisting of a chemokine and at least a fragment of achemokine receptor protein is capable of affecting immune systemfunctioning. The inventors have found that a combination consisting of achemokine and at least a fragment of a chemokine receptor can affectimmune system function in a way that is different from the effectobserved when the chemokine or at least, a fragment of the chemokinereceptor is administered alone. The pharmaceutical composition of theinvention may be used to alter immune system functioning, and thus totreat immune system diseases.

Thus, in its first aspect, the present invention provides apharmaceutical composition comprising:

-   -   (a) A cytokine;    -   (b) A peptide comprising at least a portion of a cytokine        receptor or aG-protein coupled receptor (GPCR); and    -   (c) A physiologically acceptable carrier. The cytokine may be a        chemokine.

If the receptor may is a cytokine receptor it may be a chemokinereceptor.

In a preferred embodiment, the peptide of the pharmaceutical compositioncomprises at least a portion of an extracellular domain of the receptor.In an even more preferred embodiment, the peptide of the pharmaceuticalcomposition comprises at least a portion of a regulatory sequence of thereceptor. The pharmaceutical composition may include a cytokine that, inthe absence of the at least a portion of the receptor, is aninflammatory chemokine, a constitutive chemokine or a dual functionchemokine. The peptide may bind to the chemokine.

For example, the chemokine MIG, is expressed at elevated levels ininflammatory conditions and is classified as an inflammatory CK. Thepeptide WVFGNAMCK (SEQ ID NO. 5), referred to herein as “Peptide 8” is afragment of the ECL-2 of human C-C chemokine receptor type 2 (CCR2;Monocyte Chemoattractant Protein 1 receptor). As demonstrated below,Peptide 8 has a pro-inflammatory effect when administered alone todisease-induced mice, increasing inflammation by. 44% compared withdisease-induced, untreated animals. However, quite unexpectedly, acombination of MIG and Peptide 8 has an anti-inflammatory effect whenadministered to disease-induced animals.

As another example, the chemokine RANTES when administered alone todisease induced mice is pro-inflammatory, and Peptide 8 has apro-inflammatory effect when administered alone to disease-induced mice.However, a combination of RANTES and Peptide 8 when administeredtogether to disease-induced mice has an anti-inflammatory effect.

While not wishing to be bound by a particular theory, it is believedthat when the cytokine and at least portion of the receptor areadministered in a combination, the at least portion of the receptorbinds to the cytokine and thus alters the binding properties of thecytokine to receptors in vivo. Thus, in a presently preferredembodiment, the peptide binds to the cytokine.

The pharmaceutical composition may be in any form suitable foradministration. In a preferred embodiment, the pharmaceuticalcomposition is in a form suitable for injection. The peptide of thepharmaceutical composition may comprise a sequence selected from SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ IDNo. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 and SEQ ID No. 12, described below. The peptide of thepharmaceutical composition may have at least 70% homology with any oneof 30 the peptides SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No.4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12, and be capable ofbinding to the chemokine.

One or more amino acids in the peptide may be a steric enantiomer (Disomer), a rare amino acid of plant origin, an unnatural amino acid oramino acid mimetic, or a chemically modified amino acid. Such chemicallymodified amino acids are well-known in the art and include amino acidsmodified by acetylation, acylation, phosphorylation, dephosphorylation,glycosylation, myristollation, amidation, aspartic acid/asparaginehydroxylation, phosphopantethane attachment, methylation,methylthiolation, prensyl group attachment, intein N/C-terminalsplicing, ADP-ribosylation, bromination, citrullination, deamination,dihydroxylation, formylation, geranyl-geranilation, glycation, orpalmitoylation.

In its second aspect, the invention provides use of the pharmaceuticalcomposition of the invention for altering immune system functioning,such as an autoimmune disease, multiple sclerosis, transplant rejection,psoriasis and asthma.

In its third aspect, the invention provides a method for treating animmune system disorder comprising administering to an individual in needof such treatment a pharmaceutical composition comprising:

-   -   (a) A cytokine;    -   (b) A peptide comprising at least a portion of a cytokine        receptor or a GPCR; and    -   (c) A physiologically acceptable carrier.

In its fourth aspect, the invention provides a method for preparing apharmaceutical composition, the pharmaceutical composition comprising:

-   -   (a) a solubilized cytokine; and    -   (b) a solubilized peptide comprising at least a portion of a        cytokine receptor or a GPCR; the method comprising combining the        cytokine and the peptide ex vivo.

In its fifth aspect, the invention provides a peptide selected from:

-   -   (c) SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ        ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No.        9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12;    -   (d) a peptide having at least 70% homology with a peptide of (a)        capable of binding to a cytokine; and    -   (e) a peptide of (a) or (b) wherein at least one amino acid has        been chemically modified.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 shows chemokine binding of Chemokine Binding Peptide (CBP) 5;

FIG. 2 shows chemokine binding of CBP 8;

FIG. 3 shows chemokine binding of CBP 10;

FIGS. 4A and 4B show chemokine binding CBP 2;

FIG. 5 shows chemokine binding of CBP 7;

FIG. 6 shows a negative control micro-array analyses in the absence ofCBP5, CBP8, and CBP10 peptides (negative controls) that were performedin parallel with the binding experiment of FIGS. 1, 2 and 3,respectively;

FIG. 7 shows a negative control micro-array analyses in the absence ofCBP7, (negative control) that was performed in parallel with the bindingexperiment of FIG. 5;

FIGS. 8A and 8B show a negative control micro-array analyses in theabsence of CBP2, (negative control) that was performed in parallel withthe binding experiment of FIG. 4;

FIG. 9 shows chemokine binding of Phage-Presented Peptide (Ph-p) 11;

FIG. 10 shows chemokine binding of Ph-p13;

FIG. 11 shows chemokine binding of Ph-p15;

FIG. 12 shows chemokine binding of Ph-p16;

FIG. 13 shows chemokine binding of Ph-p17;

FIG. 14 shows chemokine binding of Ph-p18;

FIG. 15 shows chemokine binding of Ph-p20;

FIG. 16 shows the effect of CBP 8 combined with the CK, MIG whenadministered to disease, Delayed Type Hypersensitivity (DTH)-inducedmice;

FIG. 17 shows the effect of CBP 5 combined with the CK, RANTES whenadministered to disease, DTH-induced mice;

FIG. 18 shows the effect of CBP 8 combined with the CK, RANTES whenadministered to disease, DTH-induced mice;

FIG. 19 shows the effect of CBP 1 combined with the CK, MCP-1 whenadministered to disease, DTH-induced mice;

FIG. 20 shows the effect of CBP 5 combined with the CK, MCP-1 whenadministered to disease, DTH-induced mice;

FIG. 21 shows the effect of CBP-CK combinations CBP 1-RANTES combinedwith CBP 8-RANTES when administered to disease, DTH-induced mice; and

FIG. 22 shows the effect of the CBP-CK combinations, CBP 8-IP-10combined with CBP 8-RANTES when administered to disease, DTH-inducedmice.

EXAMPLES

Materials and Methods

Chemokines (CKs): the following CKs were obtained from PreproTech Inc.(Rocky Hill, N.J. USA)

Inflammatory CKs: (1) GRO-α (CXCL1 cat. no. 300-11), (2) GRO-β (CXCL2cat. no. 300-39), (3) NAP-2 (CXCL7 cat. no. 300-14), (4) IL-8 (72aa)(CXCL8 cat. no. 200-08M), (5) IL-8 (77aa) (CXCL8 cat. no. 200-08), (6)MIG (CXCL9 cat. no. 300-26), (7) IP-10 (CXCL10 cat. no. 300-12), (8)I-TAC(CXCL11 cat. no. 300-46), (9) 1-309 (CCL1 cat. no. 300-37), (10)MCP-1 (CCL2 cat. no. 300-04), (11) MCP-2 (CCL8 cat. no. 300-15), (11)MCP-4 (CCL13 cat. no. 300-24), (12) MIP-1α (CCL3 cat. no. 300-08), (13)MIP-1α (CCL4 cat. no. 300-09), (14) RANTES (CCL5 cat. no. 300-06), (15)Eotaxin (CCL11 cat. no. 300-21), (16) Eotaxin 2 (CCL24 cat. no. 300-33),(17) Eotaxin 3 (CCL26 cat. no. 300-48).

Constitutive CKs: (1) TARC (CCL17 cat. no. 300-30), (2) MDC (69aa),(CCL22 cat. no. 300-36A), (3) SDF-1α (CXCL12 cat. no. 300-28a), (4)SDF-1β (CXCL12 cat. no. 300-28b), (5) BCA-1 (CXCL13, cat. no. 300-47),(6) MIP-3α (CCL20 cat. no. 300-29A), (7) MIP-3β (CCL19 cat. no.300-29B), (8) Exodus-2 (CCL21 cat. no. 300-35), (9) TECK (CCL25 cat. no.300-45), (10) CTAC (CCL27 cat. no. 300-54).

Dual Function (Inflammatory and Constitutive) CKs: (1) Fractalkine(CX3CL1 cat. no. 300-31), (2) Lymphotactin (XCL-1 cat. no. 300-20), (3)PF-4 (CXCL4 cat. no. 300-16).

The following 13 amino acid sequences were from CKR regulatory regionswere used

SEQ ID No. 1: SYYDDVGL, referred to herein as “Peptide 1”. Origin:N-terminus of human C-C chemokine receptor type 3 (CCR3; EosinophilEotaxin receptor). The biotinylated peptide (N-terminus) was synthesizedby BiomerTechnology, USA and dissolved in DMSO (0.05% in H₂0).

SEQ ID No. 2: WVFGHGMCK, referred to herein as “Peptide 2”. Origin:Extra Cellular Loop (ECL)-2 of human C-C chemokine receptor type 3(CCR3; Eosinophil Eotaxin receptor). The biotinylated peptide(N-terminus) was synthesized by Sigma, Israel and dissolved in H₂O.

SEQ ID No. 3: LFGNDCE, referred to herein as “Peptide 5”. Origin: ECL-4of human C-C chemokine receptor type 3 (CCR3; Eosinophil Eotaxinreceptor). The biotinylated peptide (N-terminus) was synthesized bySigma, Israel and dissolved in H₂O.

SEQ ID No. 4: WVFGTFLCK, referred to herein as “Peptide 7”. Origin:ECL-2 of human C-C chemokine receptor type 2 (CCR2; MonocyteChemoattractant Protein 1 receptor). The biotinylated peptide(N-terminus) was synthesized by BiomerTechnology, USA and dissolved inDMSO (0.1% in H₂O).

SEQ ID No. 5: WVFGNAMCK, referred to herein as “Peptide 8”. Origin:ECL-2 of human C-C chemokine receptor type 2 (CCR2; MonocyteChemoattractant Protein 1 receptor). The biotinylated peptide(N-terminus) was synthesized by BiomerTechnology, USA and dissolved inDMSO (0.1% in H₂O).

SEQ ID No. 6: FFGLNNC, referred to herein as “Peptide 10”. Origin: ECL-4of human C-C chemokine receptor type 5 (CCRS; HIV-1 Fusion Co-receptor).The biotinylated peptide (N-terminus) was synthesized byBiomerTechnology, USA and dissolved in DMSO (0.1% in H₂O).

SEQ ID No. 7: TTFFDYDYG, referred to herein as “Phage-presented (Ph)Peptide 11”. Origin: N-terminus of human C-C chemokine receptor type 2(CCR2; Monocyte Chemoattractant Protein 1 receptor). The recombinantpeptide was cloned in and expressed by the M13 cloning vector.

SEQ ID No. 8: EDSVY, referred to herein as “Ph-Peptide 13”. Origin:ECL-3 of human C-C chemokine receptor type 2 (CCR2; MonocyteChemoattractant Protein 1 receptor). The recombinant peptide was clonedin and expressed by the M13 cloning vector.

SEQ ID No. 9: WVFGSGLCK, referred to herein as “Ph-Peptide 15”. Origin:ECL-2 of human C-X-C chemokine receptor type 2 (CXCR3;Interferon-inducible protein 10 receptor). The recombinant peptide wascloned in and expressed by the M13 cloning vector.

SEQ ID No. 10: HHTCSLHFP, referred to herein as “Ph-Peptide 16”. Origin:ECL-3 of human C-C chemokine receptor type 1 (CCR1; Macrophageinflammatory protein 1-alpha receptor). The recombinant peptide wascloned in and expressed by the M13 cloning vector.

SEQ ID No. 11: HYTCSSHFP, referred to herein as “Ph-Peptide 17”. Origin:ECL-3 of human C-C chemokine receptor type 5 (CCR5; HIV-1 FusionCo-receptor). The recombinant peptide was cloned in and expressed by theM13 cloning vector.

SEQ ID No. 12: DRYLNIVHAT, referred to herein as “Ph-Peptide 18”.Origin: ECL-3 of human C-X-C chemokine receptor type 3 (CXCR3;Interferon-inducible protein 10 receptor). The recombinant peptide wascloned in and expressed by the M13 cloning vector.

SEQ ID No. 13: TKCQKE, referred to herein as “Ph-Peptide 20”. Origin:ECL-3 of human C-C chemokine receptor type 2-(CCR2; MonocyteChemoattractant Protein 1 receptor). The recombinant peptide was clonedin and expressed by the M13 cloning vector.

Micro-Array Analysis of Chemokine Binding Peptides

(1) Printing: Chemokine (CK) solution (in water) was serially dilutedwith Print Reagent (GenTel BioSurfaces USA) to final concentrations of50 and 25 g/ml. BSA (Amresco, Cat. No. 0032-256)/BSA-Biotin (Sigma, Cat.No. A8549) control solutions (in water) were diluted with Print Reagentto 100 μg/ml. An automated spotting robot (16 pin print tool, 0.4 mmhead; BioRobotics, UK) was used to print the CK/BSA control, 5 repeatsper sample (25 μg/ml and 50 μg/ml), on PATH Protein Microarray Slides(GenTel, Prod. No. 2-1005/-1025) at 20°-30° C., and 50-70% relativehumidity. Printed micro-array slides were stored at Room. Temperature or4° C. for at least 24 h before use.

(2) Blocking: Block Buffer (500 μl/partition; PATHblock, GenTel, Prod.No. 2-1014) was applied to the slide for 1 hour at room temp (RT). Afterremoval of Block Buffer, the slide was air dried for 25 minutes;

(3) Peptide Binding: The peptide was applied in Wash Buffer, 300 μl(GenTel, PATH wash, Prod. No. 2-1016), per partition and incubated at RTfor 1 hour with gentle agitation;

(4) Washing: The slides were washed twice with Wash Buffer (300μl/partition);

(5) First Label Binding: The slides were incubated while protected fromdirect light with Cy3-labeled streptavidin (1 mg/ml, 300 μl/partition;DyLight 547, Pierce Prod. No. 21424) for 45 minutes at RT;

(6) First Washing: The slides were washed twice with Wash Buffer (300μl/partition);

(7) Second Label Binding: Step (5) was repeated;

(8) Second Washing: Step (6) was repeated;

(9) Rinse: the slides were rinsed twice with Rinse Buffer (PATH rinse,GenTel, Prod. No. 2-1018) then dried well;

(10) Scanning: The peptide-bound, labeled slide was scanned (LaserIntensity 60%, Gain 80%, Resolution 10 μm) with a ScanArray Life Scanner(Packard BioChip Technologies, USA);

Analysis: Quantitative analysis of micro-array was performed by theSpotReader program of Niles Scientific (USA) and presented as RelativeFluorescence (RF) as a function of CK concentration. Micro-array readout(Cy3 fluorescence) was quantified to determine the relative bindingaffinities of a CBP for the CKs and by inference, the specificity of theCBP for binding to a CK.

Micro-Array Analysis of Phage Presented Chemokine Binding Peptides

(1) Printing: Chemokine (CK) solution (in water) was serially dilutedwith Print Reagent (GenTel BioSurfaces USA) to final concentrations of50 μg and 25 μg/ml. BSA/BSA-Biotin control solutions (in water) werediluted with Print Reagent to 100 μg/ml. Amplified stock of M13 phagecontrol (M13KEgIII Cloning Vector, New England Biolabs, Cat. No. E8101S)was stored in Tris Buffered Saline (TBS; pH7.5, 4° C.) and diluted withPrint Reagent to the working titre (10⁸ pfu/μl). An automated spottingrobot (16 pin print tool, 0.4 mm head; BioRobotics, UK) was used toprint the CK/BSA control/M13 control, 5 repeats per sample (CK 25 μg/mland 50 μg/ml; BSA 100 μg/ml; M13 20 μl), on PATH Protein MicroarraySlides (GenTel, Prod. No. 2-1005/-1025) at 20°-30° C., and 50-70%relative humidity. Printed micro-array slides were stored at Room.Temperature or 4° C. for at least 24 h before use.

(2) Blocking: Block Buffer (500 μl/partition; PATHblock, GenTel, Prod.No. 2-1014) was applied to the slide for 1 hour at room temp (RT). Afterremoval of Block Buffer, the slide was air dried for 25 minutes;

(3) Phage Presented Peptide Binding: Amplified recombinant phage stockwas stored in TBS and diluted with Wash Buffer to working titer (10⁷pfu/μl). Recombinant phage suspension (300 μl) was applied per partitionand incubated at RT for 1 hour with gentle agitation.

(4) First Washing: The slides were washed twice with Wash Buffer (300μ/partition);

(5) Primary Antibody (Ab) Labeling: The slides were incubated with thePrimary Ab (1 mg/ml diluted ×2500 (Wash Reagent), 300 μl; Mouse Anti-M13monoclonal Ab; Amersham Biosciences, UK; Product Code 27-9420-01) for 45minutes at RT;

(6) Second Washing: The slides were washed twice with Wash Buffer (300μl/partition);

(7) Secondary Ab Labeling: The slides were incubated with Secondary Ab(1.5 mg/ml diluted ×5000 (Wash Reagent), 300 μl; Cy3-conjugatedAffiniPure Goat Anti-Mouse IgG; Jackson ImmunoReserch Labs, USA; ProductCode 115-165-062).

(8) Third Washing: The slides were washed twice with Wash Buffer (300μl/partition);

(9) Rinse: The slides were rinsed twice with Rinse Buffer (PATHrinse,GenTel, Prod. No. 2-1018) then dried well;

(10) Scanning: The peptide-bound, labeled slide was scanned (LaserIntensity 80%, Gain 80%, Resolution 10 μm) with a ScanArray Life Scanner(Packard BioChip Technologies, USA);

(11) Analysis: Quantitative analysis of micro-array was performed by theSpotReader program of Niles Scientific (USA) and presented as RelativeFluorescence (RF) as a function of CK concentration. Micro-array readout(Cy3 fluorescence) was quantified to determine the relative bindingaffinities of a CBP for the CKs and by inference, the specificity of theCBP for binding to a CK.

CK-Peptide Combination

Test samples which comprised specific combinations of CKs and Peptideswere prepared by mixing the CK and Peptide in a tube and storing themixture on ice for 1 hour to 3 hours before injection into anexperimental animal.

Anti-Inflammatory Control Reagent

Dexamethasone Sodium Phosphate (Dexacort Forte, Teva) was dissolved inPhosphate Buffer Saline (PBS, GIBCO) to a final concentration of 1 mg/mland injected in 2000 μl (200 μg).

The Animal Model of Disease (General Inflammation): Delayed TypeHypersensitivity

Delayed-type hypersensitivity (DTH) reactions are antigen-specific,cell-mediated immune responses that, depending on the antigen, mediatebeneficial (e.g. resistance to viruses, bacteria and fungi), or harmful(e.g. allergic dermatitis and autoimmunity) aspects of immune function.They are commonly used as models of chronic inflammatory diseases, sincethey are both initiated by an antigen and perpetuated byantigen-specific T cells.

Peptides were tested in a mouse model for their anti- orpro-inflammatory effects.

Contact hypersensitivity (CHS) reaction in mice differs from typicalskin-irritation models, in that, an agent such as oxazolone(4-ethoxymethylene-2-oxazolin-5-one) is used which is not a strongirritant and requires a sensitization exposure before challenge. Therole of oxazolone-specific T cells has been demonstrated by the abilityof purified T cells from sensitized donor mice to transfer reactivityinto naive recipients [Asherson, 1968] and both CD4⁺ and CD8+ T cellsare required to initiate the inflammatory response and recruitadditional leukocytes [Gocinski, 1990].

Sensitization: Animals (BalbC, female, age 7-8 weeks, 5 per group) weresensitized by application of oxazolone (Sigma E0753, 100 μl (2% (wt/vol)in oil (Kodak)) on abdominal skin, day 0.

Challenge: The animals were challenged by application of oxazolone (10μ1 (0.5% (wt/vol) in oil) to the left ear. 10 μl of the carrier (oil)was applied to the right ear, on day 6.

Treatment: The test reagents (CK, Peptide, CK-Peptide combinations,Anti-Inflammatory Control) were prepared from frozen stocks (−20° C.) onthe day of the treatment and stored on ice until injectedintra-peritoneally (0.2 ml per injection), one hour before Challenge andone hour after Challenge.

Measurement: Ear thickness was measured with a dial thickness gauge(Mitutoyo, Japan) twenty four hours after challenge, on day 7. The pro-and anti-inflammatory activities of individual peptides and CKs andcomplexes of peptides with CKs, were calculated relative to theanti-inflammatory activity of Dexamethasone (Dexa). Dexa is ananti-inflammatory standard which, in this example, reduced measuredinflammation by 36% and served as a standard of 100% for calculatedvalues of the pro- and anti-inflammatory activities of the testreagents. An anti-inflammatory effect was measured as a reduction ininflammation compared with the untreated group of animals and its valuecalculated relative to the efficacy of Dexa (100% anti-inflammatoryeffect). A pro-inflamatory effect was measured as an increase ininflammation compared with the untreated group and its value calculatedrelative to Dexa.

Results

Micro-array analysis of Chemokine Binding Peptide 5 (CBP5) toinflammatory, constitutively expressed and dual function CKs. FIG. 1shows the binding affinity of CBP5 to several chemokines. CBP5 boundwith relatively high affinity (>3 000 Relative Fluorescence Units (RFU),responses in the upper 75% range of 0-12000 RFUs) to the inflammatoryCKs, Eotaxin, Eotaxin 3, MCP-4 and RANTES, cognate CK ligands of thereceptor from which CBP5 is derived. The same peptide bound withrelatively high affinity to IL-8, MIG, 1309, MCP-1 and TARC, unrelatedinflammatory CK ligands of the CKR, CCR3. CBP5 also bound theconstitutively expressed CKs, SDF1-a/13, MIP3-a and TECK and the dualfunction CK, Fractalkine, none of which is a cognate ligand of the CKR,CCR3.

Micro-array analysis of Chemokine Binding Peptide 8 (CBP8) binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 2, CBP8 bound with relatively high affinity (>1500 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-6000RFUs) to the inflammatory CKs, MCP-1 and -4, cognate CK ligands of thereceptor from which CBP8 was derived. The same peptide bound withrelatively high affinity to MIG, 1-309, Eotaxin, Eotaxin 3, and to theconstitutively expressed CKs, SDF1-(3, BCA-1, Exodus-2, TECK, and thedual function CK, Fractalkine, all of which are unrelated CK ligands ofthe CKR from which CBP8 is derived.

Micro-array analysis of Chemokine Binding Peptide 10 (CBP10) binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 3, CBP10 bound with relatively high affinity (>5000 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-20000RFUs) to the inflammatory CK, MCP2, cognate ligand of the CKR from whichCBP10 is derived. The same peptide bound with relatively high affinityto the inflammatory CKs, GRO-β, IP-10, I-TAC, 1-309, MCP-1, -4, Eotaxin,Eotaxin 2/3 and to the constitutively expressed CKs, SDF1-α/1β BCA-1Exodus-2 and TECK, and the dual function CKs, Fractalkine andLymphotactin, all of which are unrelated CK ligands of the CKR fromwhich CBP10 is derived.

CBP2 (WVFGHGMCK (SEQ ID NO. 2); from the Extra Cellular Loop (ECL)-2 ofhuman C-C CKR type 3 (CCR3)) CBP2 was screened against the humaninflammatory CKs, IL-8 (72aa; CXCL8), MIG (CXCL9), 1-309 (CCL1), MCP-1(CCL2), MIP-1α (CCL3). RANTES (CCL5), (15) Eotaxin (CCL11), Eotaxin 2(CCL24), Eotaxin 3 (CCL26), TARC (CCL17), the human constitutive CKs,SDF-1α (CXCL12), SDF-1β (CXCL12), MIP-3α (CCL20) and the CK, PF-4(CXCL4). The results are shown in FIGS. 4A/B. Experimental readout (Cy3fluorescence) was quantified to determine the relative bindingaffinities of CBPs for the CKs and by inference, the binding specificityof CBPs for the CKs. CBP2.

bound with relatively high affinity (>10000 Relative Fluorescence Units(RFU), responses in the upper 75% range of 0-60000 RFUs) to theinflammatory CKs, Eotaxin and Eotaxin 3, cognate CK ligands of thereceptor from which CBP2 is derived. The same peptide bound withrelatively high affinity to the inflammatory CKs, MIG and 1-309 and theconstitutively expressed CKs, SDF1-α/β and MI P3-α, all of which areunrelated CK ligands of the CKR from which CBP2 is derived.

CBP2, derived from ECL-2 of the CKR, CCR3, bound with relatively highaffinity to Eotaxin 3, cognate CK ligand of the receptor. CBP2interaction with Eotaxin 3 is consistent with the physiologicalinteraction of a CK with its cognate receptor. The same peptide boundwith relatively high affinity to MIG and 1-309, unrelated inflammatoryCK ligands of the CKR, CCR3. CBP2 also bound the constitutivelyexpressed CKs, SDF1-α/β and MIP3-α, none of which is a cognate ligand ofthe CKR, CCR3. It is inferred from the latter observation that theunrelated inflammatory and constitutive CKs modify CCR2 activity byinteracting with CBP2 sequence, in the structural context of the CKR,CCR3. Independent of its native CKR, CBP3 is a potential modulator of CKactivity, that of cognate and unrelated CK ligands of the CKR, CCR3.

CBP7 (WVFGTFLCK (SEQ ID NO. 4); from ECL-2 of human C-C chemokinereceptor type 2 (CCR2) Peptide 7 (CBP7) was screened against the humaninflammatory CKs, IL-8 (72aa; CXCL8), MIG (CXCL9), IP-10 (CXCL10), I-TAC(CXCL11), 1-309 (CCL1), MCP-1 (CCL2), MIP-1α (CCL3), RANTES (CCL5),Eotaxin (CCL11), Eotaxin 2 (CCL24), Eotaxin 3 (CCL26), TARC (CCL17), thehuman constitutive CKs, SDF-1α (CXCL12), (CXCL12), MIP-3α (CCL20) andthe CK of undefined function, PF-4 (CXCL4) presented in micro-arrayformat. The experimental readout (Cy3 fluorescence) was quantified todetermine the relative binding affinities of the CBP for the CKs and byinference, the binding specificity of CBP7 for the CKs.

The results are shown in FIG. 5. CBP7 bound with relatively highaffinity (>2000 Relative Fluorescence Units (RFU), responses in theupper 75% range of 0-10000 RFUs) to the inflammatory CKs 1-309, Eotaxinand Eotaxin 3 and to the constitutively expressed CKs, SDF1-α. and -β,none of which is a cognate CK ligand of the CKR from which CBP7 isderived.

Negative Control Micro-array analyses for Chemokine Binding Peptides toinflammatory, constitutively expressed and dual function CKs.Micro-array analyses in the absence of peptide (negative controls) wereperformed in parallel with analyses in the presence of peptide. Negativecontrol results for CBP5, -8, and 10 are shown in FIG. 6. Negativecontrol results for CBP7 are shown in FIG. 7. Negative control resultsfor CBP2 are shown in FIGS. 5A and 8B.

Micro-array analysis of Phage-Presented Peptide, Ph-pH, binding to 30inflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 9, Ph-p11 bound with relatively high affinity (>5000 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-20000RFUs), to the inflammatory CKs, MCP-1, -2 and -4, cognate CK ligands ofthe receptor from which the peptide of Ph-p11 was derived. The samepeptide bound with relatively high affinity to the inflammatory CKs,GRO-β, MIG, RANTES, Eotaxin, Eotaxin 3 and to the constitutivelyexpressed CK, TECK, and the dual function Lymphotactin, all of which areunrelated CK ligands of the CKR from which the peptide of Ph-p 11 isderived. The RFU values of the recombinant phage were calculated bydeducting the RFU value of the phage by itself (negative control).

Micro-array analysis of Phage-Presented Peptide, Ph-p13, binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 10, Ph-p13 bound with relatively high affinity (>1500 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-6000RFUs) to the inflammatory CK, MCP-4, cognate CK ligand of the receptorfrom which the peptide of Ph-p13 was derived. The same peptide boundwith relatively high affinity to the inflammatory CKs, GRO-β, Eotaxin,Eotaxin 3 and to the constitutively expressed CK, TECK, all of which areunrelated CK ligands of the CKR from which the peptide of Ph-p13 isderived. The RFU values of the recombinant phage were calculated bydeducting the RFU value of the phage by itself (negative control).

Micro-array analysis of Phage-Presented Peptide, Ph-p15, binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 11, Ph-p15 bound with relatively high affinity (>30000 RelativeFluorescence Units (RFU), responses in the upper 50% range of 0-60000RFUs) to the inflammatory CKs, MIG, IP-10 and I-TAC, cognate CK ligandsof the receptor from which the peptide of Ph-p 15 was derived. The samepeptide bound with relatively high affinity to the inflammatory CKs,GRO-β, IL-8 (77), MCP1/2/4, RANTES, Eotaxin, Eotaxin 2, Eotaxin 3 and tothe constitutively expressed CKs, BCA-1, Exodus 2 and TECK and the dualfunction CKs, Fractalkine and Lymphotactin, all of which are unrelatedCK ligands of the CKR from which the peptide of Ph-p 15 is derived. TheRFU values of the recombinant phage were calculated by deducting the RFUvalue of the phage by itself (negative control).

Micro-array analysis of Phage-Presented Peptide, Ph-p16, binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 12, Ph-p16 bound with relatively high affinity (>30000 RelativeFluorescence Units (RFU), responses in the upper 50% range of 0-60000RFUs) to the inflammatory CKs, MCP-4 and RANTES, cognate CK ligands ofthe receptor from which the peptide of Ph-p 16 was derived. The samepeptide bound with relatively high affinity to the inflammatory CKs,GRO-β, MIG, MCP1/2, Eotaxin, Eotaxin 2, Eotaxin 3 and to theconstitutively expressed CKs, BCA-1, Exodus 2 and TECK and the dualfunction CKs, Fractalkine and Lymphotactin, all of which are unrelatedCK ligands of the CKR from which the peptide of Ph-p16 is derived. TheRFU values of the recombinant phage were calculated by deducting the RFUvalue of the phage by itself (negative control).

Micro-array analysis of Phage-Presented Peptide, Ph-p17, binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 13, Ph-p17 bound with relatively high affinity (>4500 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-22000RFUs) to the inflammatory CKs, MCP-2 and RANTES, cognate CK ligands ofthe receptor from which the peptide of Ph-p 17 was derived. The samepeptide bound with relatively high affinity to the inflammatory CKs,GRO-β, IL-8 (77), MIG, IP-10, I-TAC, MCP1/4, Eotaxin, Eotaxin 2 andEotaxin 3 and to the constitutively expressed CKs, BCA-1, Exodus 2 andTECK and the dual function CK Lymphotactin, all of which are unrelatedCK ligands of the CKR from which the peptide of Ph-p17 is derived. TheRFU values of the recombinant phage were calculated by deducting the RFUvalue of the phage by itself (negative control).

Micro-array analysis of Phage-Presented Peptide, Ph-p18, binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 14, Ph-p18 bound with relatively high affinity (>6500 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-26000RFUs) to the inflammatory CKs, MIG and 1-TAC cognate CK ligands of thereceptor from which the peptide of Ph-p18 was derived. The same peptidebound with relatively high affinity to the inflammatory CKs, GRO-β, IL-8(72), IL-8 (77), MCP1/2/4, RANTES, Eotaxin, Eotaxin 2, Eotaxin 3 and tothe constitutively expressed CKs, BCA-1, Exodus 2 and TECK and the dualfunction CKs, Fractalkine and Lymphotactin, all of which are unrelatedCK ligands of the CKR from which the peptide of Ph-p18 is derived. TheRFU values of the recombinant phage were calculated by deducting the RFUvalue of the phage by itself (negative control).

Micro-array analysis of Phage-Presented Peptide, Ph-p20, binding toinflammatory, constitutively expressed and dual functions CKs. As shownin FIG. 15, Ph-p20 bound with relatively high affinity (>12500 RelativeFluorescence Units (RFU), responses in the upper 75% range of 0-50000RFUs) to the inflammatory CKs, MCP-1, -2 and -4, cognate CK ligands ofthe receptor from which the peptide of Ph-p20 was derived. The samepeptide bound with relatively high affinity to the inflammatory CKs,GRO-β, IL-8 (72), IL-8 (77), IP-10, I-TAC, RANTES, Eotaxin, Eotaxin 3and to the constitutively expressed CKs, BCA-1, Exodus 2 and TECK andthe dual function CKs, Fractalkine and Lymphotactin, all of which areunrelated CK ligands of the CKR from which the peptide of Ph-p20 isderived. The RFU values of the recombinant phage were calculated bydeducting the RFU value of the phage by itself (negative control).

Efficacy of the CK MIG (CXCL9), Peptide 8 and different doses of Peptide8 combined with MIG. The CK MIG, is expressed at elevated levels ininflammatory conditions and defined as an inflammatory CK. As shown inFIG. 16, 200 ng MIG, administered to disease induced mice waspro-inflammatory, increasing inflammation by 38% (−38 in FIG. 16)compared with disease-induced, untreated animals. 1100 ng Peptide 8 hada pro-inflammatory effect when administered to disease-induced mice,increasing inflammation by 44% (−44) compared with disease-induced,untreated animals. A combination of MIG and Peptide 8 had ananti-inflammatory effect when administered to the disease-inducedanimals. With a dose consisting of 20 ng MIG and 100 ng Peptide 8, theanti-inflammatory effect was 5% (5), compared with disease-induced,untreated animals. With a dose consisting of 10 Ong MIG: and 550 ngPeptide 8, and with a dose consisting of 200 ng MIG and 1000 ng Peptide8, the anti-inflammatory effect was 55% (55) and 14% (14), respectively.The molecular ratio of the respective doses was 1:50 (CK:Peptide).

Efficacy of the CK, RANTES (CCL5) and Peptide 5 and different doses ofPeptide 5 combined with RANTES The CK RANTES, is expressed at elevatedlevels in inflammatory conditions and is classified as an inflammatoryCK. As shown in FIG. 17, 200 ng RANTES, administered to disease inducedmice was slightly pro-inflammatory, increasing inflammation by <3% (<−3in FIG. 17) compared with disease-induced, untreated animals. 1,300 ngPeptide 5 had an anti-inflammatory effect when administered todisease-induced mice, decreasing inflammation by 11% (11) compared withdisease-induced, untreated animals. A combination of RANTES and Peptide5 had a pro-inflammatory effect of 18% (−18) when administered to thedisease-induced animals at a dose consisting of 2 Ong RANTES and 13 OngPeptide 5. At a dose consisting of 10 Ong RANTES and 550 ng Peptide 5,the anti-inflammatory effect of the combination was 34% (34), comparedwith disease-induced, untreated animals. At a dose consisting of 200 ngRANTES and 1300 ng Peptide 5, the anti-inflammatory effect was 18% (18).The molecular ratio of the respective doses was 1:50 (CK:Peptide). Giventhe pro-inflammatory-activity of RANTES alone and the anti-inflammatoryactivity of Peptide 5 by itself, the activities of the CK-Peptidecombinations are evidence of formation of a RANTES-Peptide 5 complex andstability of the complex. At the sub-optimal dose (RANTES: 20 ng-Peptide5 130 ng) the complex manifested pro-inflammatory activity, consistentwith the biphasic properties of CKs. At low concentrations, CKs arechemo-attractants, inducing target (inflammatory) cell migration. Atrelatively high concentrations CKs inhibit target cell migration. Anoptimal dose (RANTES: 10 Ong-Peptide 6: 550 ng) was required for amaximal anti-inflammatory effect, consistent with the biologicalactivity of a competitive inhibitor of a disease related, wild type CK.At the supra-optimal dose, the active complex activated diseasenon-related CK receptors inducing a counter-indicative, pro-inflammatoryresponse.

Efficacy of the CK RANTES (CCL5), Peptide 8 and different doses ofPeptide 8 combined with RANTES. Referring now to FIG. 18, as shownabove, 200 ng of the CK RANTES when administered to disease induced miceis pro-inflammatory, increasing inflammation by <3% (<−3 in FIG. 18)compared with disease-induced, untreated animals. 1100 ng Peptide 8 hada pro-inflammatory effect when administered to disease-induced mice,increasing inflammation by 44% (−44) compared with disease-induced,untreated animals. A combination of RANTES and Peptide 8 had ananti-inflammatory effect when administered to the disease-inducedanimals at two doses, RANTES: 2 ng and Peptide 8: 20 ng (52% (52)) andRANTES: I0 ng and Peptide 8: 100 ng (25% (25)). With a dose of RANTES:50 ng and Peptide 8: 500 ng, the pro-inflammatory effect was 36% (−36),compared with disease-induced, untreated animals. The molecular ratio ofthe respective doses was 1:50 (CK:Peptide). The pro-inflammatoryactivities of the individual components (RANTES and Peptide 8) and theanti-inflammatory activities of the CK-Peptide combinations are evidenceof formation of a stable complex. Maximal anti-inflammatory activity wasobserved with combination consisting of RANTES: 2 ng and Peptide 8: 20ng, consistent with the biological activity of a competitive inhibitorof a disease related, wild type CK. At the supra-optimal doses, theactive complex activated disease non-related CK receptors inducing acounter-indicative, pro-inflammatory response.

Efficacy of the CK MCP1 (CCL2), Peptide 1 and different doses of Peptide1 combined with MCP1. The CK MCP1, is expressed at elevated levels ininflammatory conditions and is classified as an inflammatory CK. Asshown in FIG. 19, 200 ng MCP1, administered to disease induced mice, waspro-inflammatory, increasing inflammation by 23% (−23 in FIG. 19)compared with disease-induced, untreated animals. 1,400 ng Peptide 1 hada pro-inflammatory effect when administered to disease-induced mice,increasing inflammation by 34% (−34) compared with disease-induced,untreated animals. A combination of MCP 1 and Peptide 1 had apro-inflammatory effect when administered to the disease-induced animalsat two doses, MCP1: 20 ng and Peptide 1: 140 ng (55% (55)) and MCP1: 100ng and Peptide 1: 700 ng (23% (23)). At a dose of MCP1: 200 ng andPeptide 1: 1400 ng, the anti-inflammatory effect was 33% (33), comparedwith disease-induced, untreated animals. The molecular ratio of therespective doses was 1:50, CK: Peptide. The pro-inflammatory activitiesof the individual components (MIG and Peptide 1) and theanti-inflammatory activity of the CK-Peptide combination is evidence offormation of a stable complex. At the sub-optimal doses (MCP1: 20ng-Peptide 1: 140 ng; MCP1: 100 ng-Peptide 1: 700 ng) the complexmanifested pro-inflammatory activity, consistent with the biphasicproperties of CKs. At low concentrations CKs are chemo-attractants,inducing target (inflammatory) cell migration. At relatively highconcentrations CKs inhibit target cell migration. An optimal dose (MCP1: 200 ng and Peptide 1: 1400 ng was required for the anti-inflammatoryeffect, consistent with the biological activity of a competitiveinhibitor of a disease related, wild type CK.

Efficacy of the CK, MCP1 (CCL2), Peptide 5 and different doses ofPeptide 5 combined with MCP1. The CK MCP1, is expressed at elevatedlevels in inflammatory conditions and is classified as an inflammatoryCK. As shown in FIG. 20, 200 ng MCP1, administered to disease inducedmice was pro-inflammatory, increasing inflammation by 23% (−23 in FIG.20) compared with disease-induced, untreated animals. 1300 ng Peptide 5had an anti-inflammatory effect when administered to disease-inducedmice, decreasing inflammation by 11% (11) compared with disease-induced,untreated animals. A combination of MCP1 and Peptide 5 had apro-inflammatory effect when administered to the disease-induced animalsat two doses, MCP1: 20 ng and Peptide 5: 120 ng (23% (23)) and MCP1: 200ng and Peptide 5: 1200 ng (11% (−11)). At a dose of MCP1: 100 ng andPeptide 5: 600 ng the anti-inflammatory effect was 79% (79), comparedwith disease-induced, untreated animals. The molecular ratio of therespective doses was 1:50, CK: Peptide. The pro-inflammatory activitiesof the individual components (MCP1 and Peptide 5) and theanti-inflammatory activity of the CK-Peptide combination is evidence offormation of a stable complex. At a sub-optimal dose (MCP 1: 20ng-Peptide 5: 120 ng) the complex manifested pro-inflammatory activity,consistent with the biphasic properties of CKs. At low concentrations,CKs are chemo-attractants, inducing target (inflammatory) cellmigration. At relatively high concentrations CKs inhibit target cellmigration. An optimal dose (MCP1: 100 ng and Peptide 5: 600 ng) wasrequired for the anti-inflammatory effect, consistent with thebiological activity of a competitive inhibitor of a disease related,wild type CK. At the supra-optimal dose, the active complex activateddisease non-related CK receptors inducing a counter-indicative,pro-inflammatory response.

Efficacies of the CK-Peptide combinations, RANTES (CCL5)-Peptide 1 andRANTES-Peptide 8, separately and together. As shown in FIG. 21, a doseconsisting of a combination of 100 ng RANTES and 750 ng Peptide 1,administered to disease induced mice, was pro-inflammatory, increasinginflammation by 30% (−30 in FIG. 21) compared with disease-induced,untreated animals. In contrast to this, a dose consisting of acombination of 10 ng RANTES and 100 ng Peptide 8 was anti-inflammatory,decreasing inflammation by 25% (25). The counter-indicative therapeuticactivities of the respective RANTES-Peptide combinations, RANTES-Peptide1 and RANTES-Peptide 8, was evidence that each of the peptides modulatedRANTES activity in an opposite way and that each combination was astable complex. The net effect of administrating RANTES-Peptide 1together with RANTES-Peptide 8 to disease-induced animals (38% (38)anti-inflammatory) has implications for the pharmacological propertiesof the complexes. The net anti-inflammatory effect may be theconsequence of competition between functionally discrete complexes forthe same disease-related CK receptor or, alternatively, the result ofactivating functionally opposed, pro- and anti-inflammatory, receptors.

Efficacies of the CK-Peptide combinations, IP10 (CXCL10)-Peptide 8 andRANTES (CCL5)-Peptide 8, separately and together. A dose consisting of100 ng IP10 and 750 ng Peptide 8, administered to the disease inducedmice, was pro-inflammatory, increasing inflammation by 60% (−60 in FIG.22) compared with disease-induced, untreated animals. A dose consistingof 10 ng RANTES and 100 ng Peptide 8 was anti-inflammatory, decreasinginflammation by 25% (25). The counter-indicative therapeutic activitiesof these two CK-Peptide combinations is evidence that Peptide 8modulated the inflammatory CK IP10 and RANTES activities in oppositeways. The net anti-inflammatory effect may be the consequence ofcompetition between functionally discrete complexes for the samedisease-related CK receptor or, alternatively, the result of activatingfunctionally opposed, pro- and anti-inflammatory, receptors.

Therapeutic Indications

Rheumatoid Arthritis: Clinical observation and empirical evidence haveindicated roles for RANTES, MIG, MCP-1 and IP10 and their respectivecognate receptors, in the pathogenesis of Rheumatoid Arthritis. Acombination consisting of RANTES and CBP 5 (see FIG. 17), RANTES and CBP8 (see FIG. 18), MIG and CBP 8 (see FIG. 16), MCP-1 and CBP 1 (see FIG.19), MCP1 and CBP 5 (see FIG. 20), or IP10 and CBP 8 (see FIG. 22), maybe used in the treatment of rheumatoid arthritis.

Asthma: The CKs, RANTES and MCP-1 and the CK receptors, CCR3, origin ofCBP 1 and CBP 5 and CCR2, origin of CBP 8, are implicated in thepathogenesis of Asthma. A combination of RANTES and CBP 5 (see FIG. 17),RANTES and CBP 8 (see FIG. 18), MCP-1 and CBP 1 (see FIG. 19), or MCP-1and CBP 5 (see FIG. 20), may be used in the treatment of Asthma.

Transplantation Rejection: The levels of CKs, MIG, RANTES, MCP-1 andIP10 and the CK receptor, CCR2, origin of CBP 8, correlate with OrganTransplant Rejection. A combination of MIG and CBP 8 (see FIG. 16),RANTES and CBP 5 (see FIG. 17), RANTES and CBP 8 (see FIG. 18), MCP-1and CBP 1 (see FIG. 19), MCP1 and CBP 5 (see FIG. 20), or IP10 and CBP 8(see FIG. 22), may be used in the treatment of Transplant Rejection.

Multiple Sclerosis: MIG, RANTES, MCP-1 and IP10 are MultipleSclerosis-related CKs and the CK receptor, CCR2, origin of CBP 8, isimplicated in the disease. A combination of MIG and CBP 8 (see FIG. 16),RANTES and CBP 5 (see FIG. 17), RANTES and CBP 8 (see FIG. 18), MCP-1and CBP 1 (see FIG. 19), MCP1 and CBP 5 (see FIG. 20), or IP10 and CBP 8(see FIG. 22), may be used in the treatment of Transplant Rejection.

Inflammatory Bowel Disease: The CK, RANTES, is implicated in thepathogenesis of Inflammatory Bowel Disease. A combination of RANTES andCBP 5 (see FIG. 17), or RANTES and CBP 8 (see FIG. 18) may be used inthe treatment of the disease.

Psoriasis: The CKs, MIG and RANTES are implicated in the pathogenesis ofPsoriasis. A combination of MIG and CBP 8 (see FIG. 16), RANTES and CBP5 (see FIG. 17), or RANTES and CBP 8 (see FIG. 18), may be used in thetreatment of the disease.

AIDS: The cognate receptor of RANTES, CCR5, is involved in HIV infectionand a validated drug target for the treatment of AIDS. A combination ofRANTES and CBP 5 (see FIG. 17), or RANTES and CBP 8 (see FIG. 18), maybe used as a competitive inhibitor of the virus in the treatment of thedisease.

CANCER: Metastasis and angiogenesis are essential for cancerpathogenesis. CKs mediate the cell migration of metastasis and thevascularization of angiogenisis. The CK-CBP combinations are potentialmodulators of and as such, therapeutic agents for, these pathogenicprocesses.

What is claimed:
 1. A method for treating an immune system disorderwherein the disorder involves delayed-type hypersensitivity reactionscomprising administering to an individual in need of such treatment apharmaceutical composition selected from the group consisting of: I. (A)a peptide selected from the group consisting of (a) the peptide of aminoacid sequence NO:5, (b) the peptide of SEQ ID NO:5 wherein the peptidehas been changed at one, two or three amino acid positions and a peptideof (a) or (b) wherein at least one amino acid has been chemicallymodified (B) a chemokine selected from the group consisting of MIG(monokine induced by gamma interferon), RANTES (C-C motif chemokine 25),MCP-1 (C-C motif chemokine 2) and IP-10 (Interferon-inducibleprotein-10) and (C) a pharmaceutically acceptable carrier; II. (A) apeptide selected from the group consisting of (a) the peptide of aminoacid sequence NO:3, (b) the peptide of SEQ ID NO:3 wherein the peptidehas been changed at one, two or three amino acid positions and a peptideof (a) or (b) wherein at least one amino acid has been chemicallymodified (B) a chemokine selected from the group consisting of RANTESand MCP-1 (C) a pharmaceutically acceptable carrier; and III. (A) apeptide selected from the group consisting of (a) the peptide of aminoacid sequence NO:1 (b) the peptide of SEQ ID NO:1 wherein the peptidehas been changed at one, two or three amino acid positions and a peptideof (a) or (b) wherein at least one amino acid has been chemicallymodified (B) a chemokine selected from the group consisting of RANTESand MCP-1 (C) a pharmaceutically acceptable carrier.
 2. The methodaccording to claim 1, wherein the composition is in a form suitable forinjection.
 3. The method according to claim 1 wherein the peptide bindsto the chemokine.
 4. The method according to claim 1 wherein thechemokine is an inflammatory chemokine.
 5. The method according to claim1 wherein the chemical modification is selected from the groupconsisting of acetylation, acylation, phosphorylation,dephosphorylation, glycosylation, myristollation, amidation, asparticacid/asparagine hydroxylation, phosphopantethane attachment,methylation, methylthiolation, prensyl group attachment, inteinN-/C-terminal splicing, ADP-ribosylation, bromination, citrullination,deamination, dihydroxylation, formylation, geranyl-geranilation,glycation, and palmitoylation.